Fawell S E, Lenard J
Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
Biochem Biophys Res Commun. 1988 Aug 30;155(1):59-65. doi: 10.1016/s0006-291x(88)81049-0.
Cells of the wall-less ("slime") strain of Neurospora crassa possess specific high affinity insulin binding sites on their cell surface. 125I-labeled bound insulin was not displaced from these cells by insulin-like growth factor II (IGF-II), and was only weakly displaced by IGF-I and proinsulin. Cross-linking of 125I-labeled insulin with N. crassa cells using disuccinimidyl suberate resulted in the labeling of a single band of ca. 67 kDa m.w. on a polyacrylamide gel. Two proteins of ca. 66 and 59 kDa m.w. were purified from detergent solubilized plasma membrane preparations by passage over an insulin-agarose affinity matrix. Antibodies against an autophosphorylation site on the human and Drosophila insulin receptors (anti P2) immunoprecipitated a single phosphoprotein of ca. 50 kDa m.w. from detergent solubilized plasma membranes, which possessed protein tyrosine kinase activity when histone H2 was used as substrate.
粗糙脉孢菌无细胞壁（“黏液”）菌株的细胞在其细胞表面具有特定的高亲和力胰岛素结合位点。胰岛素样生长因子II（IGF-II）不能将125I标记的结合胰岛素从这些细胞中置换出来，而IGF-I和胰岛素原只能微弱地置换它。使用辛二酸二琥珀酰亚胺酯将125I标记的胰岛素与粗糙脉孢菌细胞进行交联，在聚丙烯酰胺凝胶上产生了一条约67 kDa分子量的单一条带标记。通过胰岛素-琼脂糖亲和基质从去污剂溶解的质膜制剂中纯化出两种分子量约为66和59 kDa的蛋白质。针对人和果蝇胰岛素受体上的自磷酸化位点的抗体（抗P2）从去污剂溶解的质膜中免疫沉淀出一种分子量约为50 kDa的单磷蛋白，当使用组蛋白H2作为底物时，该磷蛋白具有蛋白酪氨酸激酶活性。
